Pinus ponderosa, or western yellow pine (Pinaceae) is abundant in western and midwestern states and in western Canada. Needles from P. ponderosa cause cattle to abort if they are consumed during late gestation (McDonald, Veterinary Endocrinology and Reproduction 1969; Stevenson et al., Pine Needle (Pinus ponderosa)-Induced Abortion in Range Cattle, Cornell Vet., pp. 519-524, 1972). P. ponderosa ingestion induces premature parturition in cattle by causing prolonged vasoconstriction (i.e. increased vascular tone) of the caruncular arterial bed partially through increases in potential sensitive calcium channel (PSC) activity (Christenson et al., Effects of Pine Needles (Pinus ponderosa) by Late-Pregnant Beef Cows on Potential Sensitive Ca.sup.2+ Channel Activity of Caruncular Arteries, J. Reprod. Fertil. pp. 301-306, 1993) resulting in a decrease in uterine blood flow.
In pregnancy, blood flow to the gravid bovine uterus increases .apprxeq.40 fold from conception to term (Ferrell and Ford, Blood Flow, Steroid Secretion and Nutrient Uptake of the Gravid Bovine Uterus, J. Anim. Sci., 50, p. 1113-1121, 1980). After day 200, 80-85% of the uterine arterial blood flows through the caruncular arterial bed (Macowski et al., Distribution of Uterine Blood Flow in the Pregnant Sheep, Am. J. Obstet. Gynecol, 101 pp. 409-412, 1968) as a consequence of marked decreases in caruncular arterial tone (Ford, Control of Blood Flow to the Gravid Uterus of Domestic Livestock Species, J. Anim. Sci., pp. 32-43, 1994). Early calving following pine needle consumption is accompanied by a profound constriction of caruncular arteries and ischemic necrosis at the placental attachment site (Stuart, et al., Pine Needle abortion in cattle: Pathological observations, Cornell Vet. 79, pp. 61-69, 1989). It is believed that components in pine needles increase the tone of the caruncular artery, resulting in a reduction of blood flow to the fetal-maternal interface.
Decreases in uterine arterial blood flow appear to result from activation of .alpha.2-adrenergic receptors on the vascular smooth muscle membrane of the artery (Ford et al., Effects of Ponderosa pine needle ingestion on uterine vascular function in late-gestation beef cows, J. Anim. Sci., 70, pp. 1609-1614, 1992). Specifically, .alpha.2 activation facilitates extracellular uptake of calcium via PSC resulting in decreased vessel diameter (i.e. increased vessel tone) and uterine blood flow.
Pinus ponderosa is the only known species of Pinus to cause abortion in cattle in the United States and Canada (Pammel, Manual of Poisonous Plants, The Torch Press p. 330, 1911; James et al., Pine Needle Abortion in Cattle: A Review and Report of Recent Research, Cornell Vet., pp. 39-52, 1989; Allison and Kitts, Further studies on the anti-estrogenic activity of yellow pine needles, J. Anim. Sci., pp. 1155-1159, 1964). This results in large economic losses each year to the beef industry in the western United States (Lacey et al., Ponderosa Pine: Economic Impact In: The ecology and economic impact of poisonous plants on livestock production, pp. 95-106, 1988).
Both green and dry needles appear to cause abortion (Jensen et al., Evaluation of Histopathologic and Physiologic Changes in Cows Having Premature Births After Consuming Ponderosa Pine Needles, Am. J. Vet. Res. pp. 285-289, 1989), and bark and branch tips appear to contain abortifacient principles (Panter et al., Premature Bovine Parturition Induced by Ponderosa Pine: Effects of Pine Needles, Bark, and Branch Tips, Cornell Vet., pp. 329-338, 1990). The nature of the abortifacient principles of ponderosa pine needles has been sought for nearly forty years. Many test animals or organ assays have been used to suggest a number of substances ranging from luteolytic agents like the prostaglandins to mycotoxins or the presence of infectious microorganisms (James et al., Pine Needle Abortion in Cattle: A Review and Report of Recent Research, Cornell Vet. 79, pp. 39-52 1989).
James and coworkers recently reported that pine needles extracted with selected solvents lost their ability to induce parturition when fed to pregnant cows (James et al., Effects of Feeding Ponderosa Pine Needle Extracts and Their Residues to Pregnant Cattle, Cornell Vet. pp. 33-39, 1994). The diterpene known as isocupressic acid was previously isolated from P. ponderosa (Zinkel and Magee, Resin Acids of Pinus ponderosa Needles, Phytochemistry, pp. 845-848, 1991), and diterpene resins have been implicated in embryotoxic effects in mice (Kubic and Jackson, Embryo Resorption in Mice Induced by Diterpine Resin Acids of Pinus ponderosa Needles, Cornell Vet. pp. 34-42, 1981).
James et al., found that an 80% pure sample of isocupressic acid isolated from P. ponderosa needles and bark induced early parturition in pregnant cattle (Gardner et al., Ponderosa Pine Needle-Induced Abortion in Beef Cattle: Identification of Isocupressic Acid as the Principle Active Compound. J. Agric. Food Chem. pp. 756-761, 1994) thus identifying one abortifacient principle in ponderosa pine needles. To date, isocupressic acid is the only compound that has been isolated and successfully shown to cause abortion in pregnant beef cattle.
Numerous classes of natural products have been isolated from P. ponderosa including volatile monoterpenes, sesquiterpenes, diterpenes, and wax alcohols and acids from chitin and suberin. It has now been discovered that the lipid diester component from P. ponderosa increases caruncular arterial tone partially through increasing PSC activity. These lipids may play a role in causing early parturition in pregnant beef cattle and may potentially be used to cause parturition in other mammals as well.
Accordingly, it is a primary objective of the present invention to provide a process for preparing novel vasoactive lipid substances which induce parturition in mammals.
Another primary objective of the present invention is to prepare a range of esterified fatty acids which can be investigated as drugs for the inducement of parturition.
A still further objective of the present invention is to provide a wide range of esterified fatty acids which can be systematically used and tested to determine structure-activity relationships for increasing caruncular arterial tone partially through increase in PSC activity.